Target-selected inactivation of the zebrafish rag1 gene

The zebrafish has become a favorite organism for genetic analysis of vertebrate development, but methods for generating mutants by reverse genetic approaches have been lacking. We report a method to obtain stable mutants of a gene based on knowledge of the gene sequence only. Parental fish were mutagenized with N-ethyl-N-nitrosourea; in 2679 [F.sub.1] fish, the rag1 gene was analyzed for heterozygous mutations by resequencing. In total, we found 15 mutations: 9 resulted in amino acid substitutions and 1 resulted in a premature stop codon. This truncation mutant was found to be homozygous viable and defective in V(D)J joining. Although presumably immune deficient, these homozygous rag1 mutant fish are able to reach adulthood and are fertile. As sperm samples from all 2679 [F.sub.1] fish were collected and cryopreserved, we have in principle generated a mutant library from which mutants of most zebrafish genes can be isolated.
Forward genetic screens in the zebrafish have identified thousands of mutants defective in many kinds of biological processes (1, 2). Cloning of the affected genes is accelerated by both an [...]